Recent discoveries have established that the Na,K-ATPase, in addition to its well known role as an ion pump, serves as an important hormone receptor for endogenous cardiotonic steroid (CTS) hormones. CTSs interact with the highly conserved CTS receptor on the Na,K-ATPase to regulate renal salt handling, blood pressure, and tissue remodeling in the kidney, heart and vasculature. Circulating levels of cardiotonic steroids are elevated in a wide range of hypertensive disorders and correlate directly with blood pressure. Chronically elevated blood pressure leads to kidney remodeling and renal failure. This proposal addresses two critical gaps in the field: 1) only a few of the cardiotonic steroid (CTS) hormones which mediate these actions in vivo have been structurally identified; and 2) the role of the tissue stores of cardiotonic steroids is completely unknown, although they comprise > 99% of the total CTS content in mammals. Specific Aim 1 is to purify and structurally characterize a novel mammalian cardiotonic hormone obtained from kidney. The hypotheses is that novel cardiotonic hormones are present in mammals and have distinct chemical structures which underlie their diverse actions in the kidney, heart and vasculature. This compound will be purified using an innovative, enzyme receptor affinity-based method, and its structure will be characterized using high field-strength cryo-NMR, in collaboration with the National Magnetic Resonance Facility at Madison. Specific Aim 2 is to investigate the physiological role of the tissue stores of cardiotonic hormone in the acute regulation of blood pressure. Other studies have shown that stress or exercise produces large, acute changes in the plasma concentration of CTSs. This demonstrates that CTSs can behave as a rapidly regulated hormone in response to altered demand. Notably, the exercise stressor increase in plasma CTS is hundreds fold greater than the steady elevation found in chronic hypertension and it correlates directly with acute changes in blood pressure. This increase is too rapid and large to be accounted for by the content of CTS in adrenals, and suggests that additional storage and release sites must exist. The hypothesis for Aim 2 is that the tissue stores of cardiotonic steroids contribute to blood pressure regulation in response to acute stressors. The approach will be to measure blood pressure and plasma concentrations of cardiotonic steroids during physical exercise or evoked muscle contraction, in wild-type and gene targeted mice having depleted tissue stores due to altered affinity or expression of its receptor on Na,K-ATPase. Overall, this project will provide the definitive structure of one novel mammalian hormone from kidney, and a more complete understanding of the role of the tissue stores of CTS hormones in acute blood pressure regulation. This contribution will have an important impact because it will lead to new ways of thinking about the cardiotonic hormones and their receptors in kidney disease and blood pressure regulation, and enable future studies of their potential as therapeutic targets for hypertension.